Relationships between cystic fibrosis transmembrane conductance regulator, extracellular nucleotides and cystic fibrosis

s 112 (2006) 719–732www.elsevier.com/locate/pharmthera

Pharmacology & Therapeutic

Relationships between cystic fibrosis transmembrane conductance regulator,extracellular nucleotides and cystic fibrosis☆

Brice Marcet a,⁎, Jean-Marie Boeynaems a,b

a Institute of Interdisciplinary Research, IRIBHM, Université Libre de Bruxelles, Campus Erasme (Bât C5-110), route de Lennik 808, 1070 Brussels, Belgiumb Department of Medical Chemistry, Erasme Hospital, Université Libre de Bruxelles, route de Lennik 808, 1070 Brussels, Belgium

Abstract

Cystic fibrosis (CF) is one of the most common lethal autosomal recessive genetic diseases in the Caucasian population, with a frequency ofabout 1 in 3000 livebirths. CF is due to a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTRprotein, a cyclic adenosine 5′-monophosphate (cAMP)-regulated chloride channel localized in the apical membrane of epithelial cells. CFTR is amultifunctional protein which, in addition to be a Cl-channel, is also a regulator of multiple ion channels and other proteins. In particular CFTRhas been reported to play a role in the outflow of adenosine 5′-triphosphate (ATP) from cells, but this remains controversial. Extracellularnucleotides are signaling molecules that regulate ion transport and mucociliary clearance by acting on P2 nucleotide receptors, in particular theP2Y2 receptor. Nucleotides activating the P2Y2 receptor represent thus one pharmacotherapeutic strategy to treat CF disease, via improvement ofmucus hydration and mucociliary clearance in airways. Phase II clinical trials have recently shown that aerosolized denufosol (INS37217,Inspire®) improves pulmonary function in CF patients: denufosol was granted orphan drug status and phase III trials are planned. Here, we reviewwhat is known about the relationship between extracellular nucleotides and CFTR, the role of extracellular nucleotides in epithelialpathophysiology and their putative role as therapeutic agents.© 2006 Elsevier Inc. All rights reserved.

Keywords: Cystic fibrosis; CFTR; Chloride channels; Extracellular nucleotides; P2Y receptors; Airway epithelium

Abbreviations: ABC, adenosine 5′-triphosphate-binding cassette; ADO, adenosine; ADP, adenosine 5′-diphosphate; AEC, airway epithelial cell; AMP, adenosine 5′-monophosphate; ASL, airway surface liquid; ATP, adenosine 5′-triphosphate; ATPase, adenosine triphosphatase; CaCC, Ca2+-activated chloride channel; cAMP,cyclic adenosine 5′-monophosphate; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CHO, Chinese hamster ovary; ClC, chloridechannels; DC, dendritic cell; ENaC, epithelial sodium channel; IDO, indoleamine 2,3-dioxygenase; mRNA, messenger ribonucleic acid; NBD, nucleotide-bindingdomain; NSAP, nonspecific alkaline phosphatase; ORCC, outwardly rectifying chloride channel; PCL, periciliary liquid; PKA, protein kinase A; PKC, protein kinaseC; RNA, ribonucleic acid; SLPI, secretory leukocyte protease inhibitor; UDP, uridine 5′-diphosphate; UTP, uridine 5′-triphosphate.

☆ The author⁎ CorrespondE-mail add

0163-7258/$ -doi:10.1016/j.p

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7202. Does cystic fibrosis transmembrane conductance regulator control adenosine 5′-triphosphate

release? A controversial issue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7203. Role of P2 receptors in the regulation of cystic fibrosis transmembrane conductance

regulator and other chloride channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7223.1. Cystic fibrosis transmembrane conductance regulator and P2Y1 receptor. . . . . . . . . . . . . 7223.2. Cystic fibrosis transmembrane conductance regulator, chloride channels and P2Y2 receptors . . 7223.3. Cystic fibrosis transmembrane conductance regulator, P2Y4 and P2Y6 receptors . . . . . . . . . 7233.4. Cystic fibrosis transmembrane conductance regulator and P2X receptors. . . . . . . . . . . . . 724

s attest that there is no conflicts of interest.ing author.resses: [email protected], [email protected] (B. Marcet).

see front matter © 2006 Elsevier Inc. All rights reserved.harmthera.2006.05.010

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http://dx.doi.org/10.1016/j.pharmthera.2006.05.010

720 B. Marcet, J.-M. Boeynaems / Pharmacology & Therapeutics 112 (2006) 719–732

4. Physiological role of extracellular nucleotides in epithelia . . . . . . . . . . . . . . . . . . . . . . . . 7245. Extracellular nucleotides as therapeutic agents in cystic fibrosis . . . . . . . . . . . . . . . . . . . . . 7256. Airway inflammation in cystic fibrosis and its potential modulation by nucleotides . . . . . . . . . . . 7267. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728

1. Introduction

This review provides an update on the current understandingof the relationship between cystic fibrosis transmembraneconductance regulator (CFTR) and extracellular nucleotidesand the use of nucleotides as therapeutic agents for treatingcystic fibrosis (CF) patients. We discuss published data,preliminary data and assumptions that concern the interactionbetween CFTR and extracellular nucleotides.

CF is one of the most common lethal autosomal recessivegenetic diseases caused by mutations in CFTR (Riordan et al.,1989), a cyclic adenosine 5′-monophosphate (cAMP)-regulatedchloride channel (ClC) localized in the apical membrane ofepithelial cells (Cheng et al., 1991). Two different hypothesesusing known properties of CFTR has emerged to attempt toexplain lung CF pathogenesis: the “low volume hypothesis” andthe “high salt hypothesis” (for review, see Wine, 1999).Interestingly, both hypotheses consider CFTR's role in saltabsorption to be primary. According to the classical view, called“low volume hypothesis” (Matsui et al., 1998; for review, seeWine, 1999), dysfunction in CFTR activity leads to a defect inCl− secretion and possibly a hyperabsorption of Na+ throughthe epithelial sodium channel (ENaC) (Knowles et al., 1991;Stutts et al., 1995; Mall et al., 2000; Kunzelmann &Mall, 2001)which ultimately results in severe damage of the airways andother exocrine organs, such as pancreas and intestine. In thelungs, the CFTR defect leads to a decrease in the airway surfaceliquid (ASL) covering the epithelia and thickened mucussecretions that severely impair mucociliary clearance, thuspromoting lung infections. According to this hypothesis, bothnormal and CF ASL have plasma-like levels of salt (Matsui etal., 1998). The other theory, the “high salt hypothesis” (Smith etal., 1996; Zabner et al., 1998), postulates that normal ASL haslow levels of salt as a result of salt absorption in excess of water.According to this hypothesis, lack or dysfunction of CFTR inCF causes reduced transepithelial Cl− conductance, salt ispoorly absorbed resulting in excessively salty ASL. The highsalt level in the ASL would interfere with the bacterial killing ofnatural antibiotics such as defensins (Goldman et al., 1997) andlysozyme.

In addition to functioning as a cAMP-activated Cl-channel,CFTR could also act as a regulator of other ion channels such asoutwardly rectifying chloride channels (ORCC) (Schwiebert etal., 1995; Julien et al., 1999), Ca2+-activated chloride channels(CaCC) (Wei et al., 1999) or ENaC (Stutts et al., 1995). Severalcontroversial studies have suggested that a CFTR-dependentrelease of cellular adenosine 5′-triphosphate (ATP) (Reisin etal., 1994; Schwiebert et al., 1995; Sugita et al., 1998; Urbach &

Harvey, 1999; Walsh et al., 2000) followed by the activation ofP2 nucleotide receptors could be involved in the regulation ofthese channels by CFTR. Such an effect suggests therefore arelationship between CFTR and P2 receptor signaling.

Extracellular nucleotides are important signaling moleculesthat mediate diverse biological effects, including ion transportregulation, by acting on nucleotide-gated ion channel P2X(1–7)

and G-protein coupled P2Y(1–14) receptors (for reviews, seeRalevic & Burnstock, 1998; Abbracchio et al., 2003; Leipziger,2003; Boeynaems et al., 2005). To date, 8 mammalian P2Yreceptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13,P2Y14) have been cloned and characterized (Lustig et al., 1993;Parr et al., 1994; Communi et al., 1995, 1997; Communi, Motteet al., 1996; Communi, Parmentier et al., 1996; Ralevic &Burnstock, 1998; Communi et al., 2001; Hollopeter et al., 2001;Abbracchio et al., 2003; Boeynaems et al., 2005). The P2Yreceptor family encompasses several subtypes: some areselective purinoceptors responsive to adenosine 5′-diphosphate(ADP: P2Y1, P2Y12, P2Y13) or ATP (P2Y11), others have aselectivity for uracil nucleotides, either uridine 5′-triphosphate(UTP: P2Y4) or uridine 5′-diphosphate (UDP: P2Y6), while theP2Y2 receptor has a mixed specificity and is activated to thesame extent by ATP and UTP. One ongoing area of researchsuggests that P2Y nucleotide receptors represent targetreceptors whose activation may have therapeutic value in CF.In particular, several studies emphasized the role of the P2Y2

receptor subtype, activated equipotently by ATP and UTP, as akey control for epithelial Cl− secretion by activating CaCC thatmay constitute a potential substitute to Cl− secretion defect inCF (Brown et al., 1991; Knowles et al., 1991; Cressman et al.,1999; Yerxa et al., 2002). According to the “low volumehypothesis”, aerosolized nucleotides have been evaluated forCF therapy as a means to increase ASL volume and to restoremucociliary clearance (see Section 5).

2. Does cystic fibrosis transmembraneconductance regulator control adenosine5′-triphosphate release? A controversial issue

CFTR is a member of the family of adenosine 5′-tripho-sphate-binding cassette (ABC) proteins that actively transport abroad range of substrates and include both eukaryotic andbacterial proteins, such as the multiple drug resistance protein(e.g., P-glycoprotein), the sulfonylurea receptor, the transporterfor antigen presentation, and the bacterial periplasmic per-meases (Riordan et al., 1989). CFTR contains 1480 amino acidsand consists of 2 homologous halves. Each half contains 6membrane-spanning segments and a nucleotide-binding domain

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(NBD). The 2 halves of CFTR are linked by a cytoplasmicregulatory domain that contains a number of consensusphosphorylation sites for multiple protein kinases (Riordan etal., 1989). CFTR is unique among ABC proteins in that itstransmembrane domains comprise an ion channel that permitsbidirectional permeation of anions rather than vectorialtransport of solutes. CFTR has been well characterized as acAMP-regulated Cl-channel localized in apical membrane ofepithelial cells (Cheng et al., 1991).

P-glycoprotein, another member of the family of ABCtransporters, mediates the electrodiffusional movement of thenucleotide ATP (Abraham et al., 1993). Amino acid sequenceand structural similarities between CFTR and P-glycoproteinhave led Cantiello et al. to study the possibility that CFTR, inaddition to functioning as a cAMP-regulated Cl-channel, couldalso transport cellular ATP. They showed in a heterologousmodel that CFTR expression leads to protein kinase A (PKA)-induced ATP currents, inhibited by CFTR Cl-channel blockers.Their data argued in favor of ATP transport directly throughCFTR channels and suggested that CFTR is at the same time aCl-channel and an ATP channel (Reisin et al., 1994). Theobservations that P-glycoprotein and CFTR are associated withATP transport have also been supported by other studies usingpatch clamp and bulk ATP measurements in systems expressingCFTR (Pasyk & Foskett, 1997; Prat et al., 1996; Cantiello et al.,1998).

As illustrated in Fig. 1, once released in the extracellularmedium by a CFTR-dependent mechanism, ATP could controlother membrane channels or transporters (Schwiebert et al.,1995, 1999; Urbach & Harvey, 1999; Walsh et al., 2000;Kunzelmann & Mall, 2001), in particular ORCC. The nameORCC is related to electrophysiological properties (outwardlyrectifying) of certain Cl− conductances, but the molecularidentity of ORCC is still unknown (Gabriel et al., 1993;Schwiebert et al., 1994). A recent study has cloned andcharacterized ClC-3B, a novel alternative splicing variant ofClC-3 (ClC-3A), belonging to the CIC gene family of Cl-channels, and that is expressed predominantly in epithelial cells.The authors proposed that ClC-3B could be a candidate for the

Fig. 1. CFTR-dependent ATP release regulates other channels. CFTR that functions avia a distinct transporter or by regulating vesicle trafficking. Extracellular nucleoabsorption via ENaC by activating the P2Y2 receptor signaling pathway. Activation

ORCC molecule (Ogura et al., 2002). Schwiebert et al. (1995)showed that CFTR activation regulated ORCC by triggering thetransport of ATP out of the cell. A combination of a single-channel, whole cell recording and Ussing chamber short-circuitcurrent recordings revealed that ATP had to cross the membranefrom cytoplasm to extracellular space to exert its regulatoryeffect on Cl− conductances. In agreement with this assumption,the addition on the extracellular side of hexokinase or apyrase,enzymes which hydrolyze ATP, abolished the activation ofORCC, whereas the activity of CFTR was maintained(Schwiebert et al., 1995).

Although it seems now that there is an established relation-ship between CFTR and ATP transport in some particular cases,the problem is to determine whether ATP is released directlythrough the pore of CFTR or indirectly through anothermechanism controlled by CFTR (for reviews, see Devidas &Guggino, 1997; Schwiebert et al., 1999). One argument againstthe direct role of CFTR channel in the transport of ATP was thatthe diameter of the molecule of ATP (∼10 Å) would be largerthan that of the pore of CFTR estimated at approximately 5.3 Å(Illek et al., 1999). However, another study estimated that thediameter of CFTR pore could be as large as 10–13 Å (Linsdell& Hanrahan, 1998), enough to be permeant to ATP. Finally,other domains of the channel such as NBD1 could also take partin the passage of molecules through the membrane (Devidas &Guggino, 1997). Other studies argue in favor of ATP release,but through a distinct ATP channel controlled by CFTR. Studiescarried out in artificial membranes showed indeed that CFTRcannot by itself transport ATP (Li et al., 1996). Transientexpression of CFTR in Madin Darby canine kidney (MDCK)cells was associated with both Cl− and ATP currents uponCFTR activation by PKA (Sugita et al., 1998). The phos-phorylation of the regulatory domain, as well as hydrolysis ofATP by the NBD of CFTR, is necessary to detect an efflux ofATP. The authors concluded that the efflux of Cl− and ATP aredependent on CFTR but have a distinct sensitivity to blockers aswell as a distinct potential of reversion. That suggested aninteraction between CFTR and a transporter of ATP whosemolecular nature still remains unknown (Sugita et al., 1998).

s a cAMP-regulated Cl-channel, modulates the ATP release, directly or indirectlytides stimulate Ca2+-dependent Cl-channels (ORCC, CaCC) and inhibit Na+

of P2Y2 receptors may also modulate CFTR activity.


Since CFTR possess a C-terminal motif known to interact withPDZ domain-containing proteins (Hall et al., 1998; Wang et al.,1998), it could be hypothesized that interaction between CFTRand putative ATP transporter might involve scaffolding proteinsand interaction with PDZ domain-containing proteins. Thedysfunction of CFTR could thus involve, in addition to thedirect defect of fluid and electrolytes transport, a defect of ATPsecretion outside the cell thus causing an additional defect ofother ion channels as well as other biological processes. Takentogether, these data suggest that CFTR does not transport ATPitself, but acts as a regulator of an associated transport systemfor ATP, a hypothesis also raised by al-Awqati (1995) andHiggins (1995).

Some other studies argue against a regulation of ATPtransport by the CFTR channel. These studies failed to detect anATP current or release of ATP dependent on CFTR withdifferent methodologies, like electrophysiological methods,radioactive ATP fluxes in reconstituted vesicles containingCFTR or intracellular calcium imaging in CFTR-expressingChinese hamster ovary (CHO) cells (Grygorczyk et al., 1996; Liet al., 1996; Reddy et al., 1996; Grygorczyk & Hanrahan, 1997;Marcet et al., 2003). An hypothesis then put forth is that therelease of ATP observed would be related to an artifact at thetime of the experiments of patch-clamp, like mechanicalstimulation of epithelial cells (Grygorczyk et al., 1996;Grygorczyk & Hanrahan, 1997; Watt et al., 1998). In asurprising way, in the same cellular model, CHO cells, someauthors detect a transport of ATP depending on the activation ofCFTR (Pasyk & Foskett, 1997; Urbach & Harvey, 1999)whereas others do not (Grygorczyk et al., 1996; Grygorczyk &Hanrahan, 1997; Marcet et al., 2003). Another explanation ofthese contradictory results obtained with the same CHO celllines would imply the presence of cofactors in the culturemedium, necessary to the transport of ATP (Schwiebert et al.,1999). Lastly, Boucher et al. did not find a difference in the levelof extracellular ATP in the airways of CF and non-CF subjects,suggesting that CFTR would not be implied in the transport ofATP in airways (Donaldson et al., 2000). To date, the role ofCFTR in ATP transport is not unambiguously demonstrated andis still debated in the literature.

3. Role of P2 receptors in theregulation of cystic fibrosis transmembraneconductance regulator and other chloride channels

Growing evidence suggests functional relationships betweenCFTR and P2Y as well as P2X receptors.

3.1. Cystic fibrosis transmembraneconductance regulator and P2Y1 receptor

Like the β2 adrenergic receptor, the P2Y1 receptor, whichis activated by ADP, and CFTR have, in their C-terminalend, a binding domain to PDZ domain-containing proteins.These proteins could act as scaffolds favoring an interactionbetween P2Y1 and CFTR in membrane microdomains (Hallet al., 1998; Naren et al., 2003; Fam et al., 2005). Recently,

we have shown that under particular conditions CFTR canmodulate the selectivity of P2Y1 receptor coupling to Gproteins (Marcet et al., 2004). We have shown that the P2Y1

receptor, which is known to couple primarily to Gq proteins,coupled to Gi/o proteins in the presence of CFTR. Thus, theexpression of CFTR in CHO-K1 cells induced a switch ofG-protein coupling selectivity of the P2Y1 receptor. Theseresults thus highlight the multipurpose role of CFTR channel(Marcet et al., 2004). In addition to this direct interactionwith CFTR, the P2Y1 receptor can modulate the activity ofboth CFTR and other Cl-channels. In Xenopus A6 cells,ADP, via P2Y1, increases both cAMP/PKA and Ca2+/proteinkinase C (PKC) activities and the PKC pathway is involvedin CFTR activation via potentiation of the PKA pathway(Guerra et al., 2004). The P2Y1 receptor is expressed in apicaland basolateral membranes of human airway epithelial cells(AEC) and its activation led to phospholipase C activation andCl− secretion, whose rate is regulated by the Ca2+-activated K+

channel (Son et al., 2004).

3.2. Cystic fibrosis transmembraneconductance regulator, chloride channels and P2Y2 receptors

In 1993–1994, the P2Y2 receptor, initially named P2U receptordue to its affinity for UTP, was cloned and characterized in mouseand human (Lustig et al., 1993, Parr et al., 1994). The P2Y2

receptor couples mainly to Gq and accessorily to Gi/o-proteins, isequipotently activated by ATP and UTP and triggers the Ca2+

signaling pathway (Ralevic & Burnstock, 1998). Earlier studieshad shown that ATP orUTP led to an increase in the Ca2+-activatedCl− secretion in normal and CF human AEC (Brown et al., 1991;Knowles et al., 1991; Mason et al., 1991). In situ hybridizationrevealed the expression of P2Y2 messenger ribonucleic acid(mRNA) in bronchial, bronchiolar and alveolar epithelia and insubmucosal gland epithelium (Yerxa et al., 2002). The study ofP2Y2-null mice has definitely demonstrated the exclusive role ofthat receptor in the stimulatory effect of ATP and UTP on trachealepithelial Cl− transport (Cressman et al., 1999). The molecularidentity of the Cl-channel involved remains to be elucidated. ATPand UTP have been shown to activate ORCC in human AEC (seeabove, Schwiebert et al., 1995) and rat tracheal epithelial cells(Hwang et al., 1996). As mentioned above, the molecular identityof ORCC remains unclear although it has been claimed that ClC-3B could be an ORCC (Ogura et al., 2002). However ORCC maynot be the only epithelial Cl-channel regulated by extracellularnucleotides. Indeed, nucleotides trigger an increase in intracellularCa2+ and may activate members of the CaCC family of Cl-channel(Mason et al., 1991;Knowles et al., 1991; Stutts et al., 1992).CaCCis the name given to Cl-channels that are activated by a rise inintracellular Ca2+. As for ORCC, themolecular identity of CaCC isstill unresolved. Recently, a new gene family of CI-channels(hClCA1–3) has been proposed to encode to CaCC (Gruber et al.,1998). Their expression may control the mucus production in non-CF and CFAEC (Toda et al., 2002; Hauber et al., 2004). However,these data remain very controversial since a study has shown thathClCA gene did not encode membrane proteins but rather solubleand secreted proteins (Gibson et al., 2005). Therefore, the


molecular identity of CaCC remains currently unresolved. Thus,nucleotides released in the extracellular environment may exerteffects on several distinct Ca2+-dependent Cl− conductances (e.g.,ORCC, CaCC, ClC, etc.).

In addition, it was recently shown that the P2Y2 receptorregulated also CFTR activity (Cantiello et al., 1994; Paradiso etal., 2001; Marcet et al., 2003). First, external ATP stimulatedhuman recombinant CFTR, transfected in C127i mouse mam-mary carcinoma cells, in a cAMP independent manner (Cantielloet al., 1994). Second, extracellular ATP orUTP could stimulate, inAEC, CFTR-dependent anion secretion through a Ca2+-indepen-dent PKC pathway subsequent to the activation of the Gq-coupledP2Y2 receptor (Paradiso et al., 2001). On the other hand, we haverecently shown an inhibitory effect of P2Y2 receptor on theactivity of CFTR channel in a heterologous model (Marcet et al.,2003). The activation of the P2Y2 receptor induced an inhibitionof the activity of CFTR by a mechanism independent of cAMP,but which could involve Ca2+ signaling, in the CFTR-expressingCHO-BQ1 cell line (Marcet et al., 2003). This action might berelated to the upregulation of CaCC which is observed in CFepithelia. Expression of CFTR has indeed been shown to inhibitCaCC-mediated current (Wei et al., 2001), while the loss of CFTRwas associated with CaCC upregulation in murine trachealepithelium (Tarran et al., 2002). Therefore it might be speculatedthat an inhibitory effect of UTP on CFTR could reinforce itsstimulatory effect on CaCC. Anyway this action should have noimpact on CF cells where CFTR is inactive.

The activation of P2Y2 receptors could also take part inthe inhibition of Na+ absorption via ENaC probably via thePKC pathway (Kunzelmann & Mall, 2001). As CF is alsocharacterized by hyperabsorption of Na+, some authors havedetermined the effect of mucosal UTP on transepithelial Na+

transport in primary cultures of human bronchial epithelia(Devor & Pilewski, 1999). They showed that UTP-inducedintracellular Ca2+ increase promoted a long-term inhibition oftransepithelial Na+ transport across normal and CF epithelialcells at least partly due to downregulation of a basolateralmembrane K+ conductance (Devor & Pilewski, 1999). Thus,UTP may have a dual therapeutic effect in CF airway bystimulating a Cl− secretory response and by inhibiting Na+

transport. However, the relative importance of an excessiveNa+ absorption in CF remains controversial (Joo et al.,2006).

All these data constitute the rationale of ongoing clinical trialsof aerosolized nucleotides in CF. In addition, because of itsexpression in the apical domain of the AEC, the P2Y2 receptorrepresents a potential target in the CF gene therapy, in order tosupport the internalisation of the vectors containing transgeneand thus to increase the efficacy of this therapy (Boucher, 1999;Kreda et al., 2000). The therapeutic role of nucleotides in CF islargely discussed in Section 5 of this review.

3.3. Cystic fibrosis transmembraneconductance regulator, P2Y4 and P2Y6 receptors

The concept that the action of nucleotides on the epithelialtransport of electrolytes and water involves multiple receptors

was confirmed by a gene targeting approach in mice. Indeed thenucleotide-stimulated Cl− secretion by the trachea was almostentirely abolished in P2Y2

−/− mice, whereas the nucleotide effectwas maintained completely in the jejunum and partially in thegallbladder (Cressman et al., 1999).

Following cloning of the murine P2Y4 gene (Suarez-Huertaet al., 2001), P2Y4-null mice were generated with aconventional gene targeting method. Sections of the mid-portion of the jejunum were mounted in Ussing chambers forbioelectric measurements. In wild-type animals, the apicalapplication of UTP increased the short circuit current. Thatresponse was abolished in P2Y4 knockout mice, whereas theyexhibited a normal response to the adenylyl cyclase activatorforskolin (Robaye et al., 2003). This work represents the firstclear-cut demonstration of a biological function of the P2Y4

receptor. It was thus demonstrated that the P2Y4 receptor isexpressed in murine jejunum and that its agonists are able tostimulate Cl− secretion in normal jejunum (Robaye et al.,2003). Similar observations were made in the colon (Ghanemet al., 2005). A response to nucleotides was also observed onthe basolateral side; that response was totally abolished in thecolon of P2Y4-null mice, whereas in the jejunum it waspartially decreased in both P2Y2- and P2Y4-deficient mice(Ghanem et al., 2005). Interestingly, the Cl− secretory responseto forskolin, mediated by CFTR, was potentiated by priorbasolateral addition of UTP and this potentiation wasabolished in P2Y4-null mice. Those data thus indicated thatthe P2Y4 receptor plays a dominant role in the gut, whereas inthe airways that role is played by the P2Y2 receptor.

In addition to the P2Y2 subtype, P2Y1, P2Y4, P2Y6, P2Y11

and P2Y12 mRNA could also be found in human airwaybronchial or nasal epithelial cells (Communi et al., 1999; Kim etal., 2004). Furthermore, there is pharmacological evidence thatin addition to the P2Y2 receptor, a functional receptorselectively responsive to UDP (presumably the P2Y6 subtype)is present on the apical surface of human nasal epithelial cells(Lazarowski et al., 1997). Recently, it was shown using Ussingchamber experiments that luminal UDP or the P2Y6 receptoragonist INS48823 induced both transient and persistentincreases in short circuit currents in the mouse trachea. Theauthors showed that activation of short circuit currents wasinhibited by the P2Y receptor blocker pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). The transientresponse was inhibited by 4,4′-diisothiocyanatostilbène-2,2′-disulfonic acid (DIDS), whereas the persistent short circuitcurrent was inhibited by glibenclamide and by PKA blockerH-89. Moreover, sustained activation of short circuit currents byluminal UDP was inhibited by blocking basolateral K+ channelswith 293B. The authors suggested that activation of luminalP2Y6 receptors in the airways shifts electrolyte transporttowards secretion by increasing intracellular Ca2+ and activa-tion of PKA (Schreiber & Kunzelmann, 2005). Finally,Leipziger et al. have shown that basolateral P2Y6 receptors incolonic epithelial cells stimulated sustained NaCl secretion byway of a synergistic increase of intracellular Ca2+ and cAMP(Kottgen et al., 2003). In support of these data, the authorsdemonstrated that in Xenopus oocytes expressing the P2Y6


receptor, UDP transiently activated the Ca2+-activated Cl−

current and subsequently CFTR (Kottgen et al., 2003).

3.4. Cystic fibrosis transmembraneconductance regulator and P2X receptors

In addition to activating P2Y receptor subtypes, extracellularATP could stimulate epithelial P2X receptors (Taylor et al.,1999; Zsembery et al., 2003). ATP-induced P2X receptoractivation promoted a sustained increase in intracellular Ca2+

concentration and potentiated Ca2+-dependant Cl− transportacross nasal mucosa of CF patients as well as across non-CFnasal mucosa. Thus, P2X receptors may provide additionaltargets for extracellular nucleotide therapy of CF (Taylor et al.,1999; Zsembery et al., 2003).

4. Physiological role of extracellular nucleotides in epithelia

The concept that adenine and uridine nucleotides function asextracellular signaling molecules has been markedly expandedin the last decade. The significance of nucleotides asextracellular molecules is also underscored by the ubiquitousdistribution of several classes of ectoenzymes that catalyzenucleotide breakdown and interconversion (Zimmermann,1996).

Normal airway epithelia are protected by a thin liquid layer:the ASL. ASL is divided into 2 layers: a mucus layer (height:∼7–70 μm in vivo) containing mucins and a periciliary liquid(PCL) layer (height: ∼7 μm), which keeps mucins at asufficient distance from the underlying epithelium to optimizecilia beating and mucus clearance (Knowles & Boucher, 2002;Tarran et al., 2006). Recent studies have demonstrated thecrucial role of external nucleotides in maintaining physiologicalASL volume (for review, see Tarran et al., 2006). IntracellularATP levels are∼5–10 mM, so relatively little cellular ATP mustbe released to reach a concentration large enough to activate P2receptors. Numerous studies showed that airway epitheliarelease ATP under basal conditions (Taylor et al., 1998;Donaldson et al., 2000) and in response to various mechanicalstimulations such as membrane stretch (Homolya et al., 2000),shear stress (Grygorczyk & Hanrahan, 1997; Watt et al., 1998;Tarran et al., 2005, 2006) and hypotonicity-induced swelling(Wang et al., 1996; Musante et al., 1999; Braunstein et al.,2001). Pathogen invasion is another mechanism that can induceATP release. Recently, it has been shown that flagellin ofPseudomonas aeruginosa inhibited ENaC by inducing ATPrelease from airway epithelia (Kunzelmann et al., 2006). ATP isalso released from tracheal epithelium by parainfluenza virus(Kunzelmann et al., 2004). UTP is co-released with ATP but inlower amounts (Lazarowski & Boucher, 2001). Lazarowski andHarden (1999) have previously described sensitive meth-odologies that allowed to measure UTP concentration. Theconcentration of UTP measured in the medium bathing ∼3·105

tissue culture cells under basal conditions ranged from 1 to5 nM and represented approximately 1/3 of the ATPconcentration. Since no difference in luminal UTP concentra-tion was found between normal and CF cells under either

resting or mechanically stimulated conditions (Lazarowski &Harden, 1999), it is also unlikely that CFTR was involved inUTP release by AEC. UDP-glucose is also released, by adistinct mechanism involving vesicle transport during traffick-ing of glycoproteins to the plasma membrane (Lazarowski et al.,2003).

Since under normal conditions, the mucus layer and the PCLare in continuous motion propelled by the ciliary beat cycle(∼40 μm/s), shear forces generated by the ASL movement mayprovide a mechanical stimulus that induces nucleotide release(Tarran et al., 2006). Moreover, bilateral release of UTP andATP may be triggered in some pathophysiological conditions inwhich the laminal PCL flow is disrupted by a mechanicalstimulus (e.g., during inhalation of foreign particles) or byincreased shear due to increased rates of airflow (e.g., cough).Although ATP and UTP concentrations measured in the dilutedmedium bathing the mucosal surface (∼200 μl/cm2) of primarycultures of resting human nasal epithelial cells seemed too lowto activate P2 receptors (Lazarowski & Harden, 1999),nucleotide levels in the thin (1 μl/cm2 or ∼20 μm of depth)film of ASL that covers the AEC surface grown at the air–liquidinterface (Jayaraman et al., 2001) might approach thresholdvalues for P2 receptor stimulation (0.1–1 μM). Furthermore theamounts released in response to mechanical stimulation mightbe sufficient to promote autocrine stimulation of P2Y receptorsas well as paracrine activation of nonepithelial P2Y and P2Xreceptors such as submucosal fibroblasts, smooth muscles andinflammatory cells (Lazarowski & Harden, 1999; Homolya etal., 2000; Tarran et al., 2006).

ATP can modulate the airway epithelium ion transport via theP2Y2 receptor but also via conversion into adenosine (ADO) byectonucleotidases. Recently, it has been shown that the ecto-adenosine triphosphatase (ATPase) activity in the mucosalsurface increased toward areas most susceptible to airwayobstruction (nose<bronchi≪bronchioles) (Picher et al., 2004).Boucher et al. have identified a nonspecific alkaline phospha-tase (NSAP) as the major ectonucleotidase in mucosal surfaceof airways. NSAP regulated nucleotide concentration in CF andwould be responsible for the termination of aerosolizednucleotide-mediated mucociliary clearance in the lungs of CFpatients (Picher et al., 2004). For example, they showed that themucosal epithelial surface eliminated P2 receptor agonists(ATP=UTP>ADP>UDP) at 3-fold higher rates than theserosal surface (Picher et al., 2004). Furthermore, the ecto-ATPase activity and mRNA expression of mucosally restrictedNSAP were 3-folds higher in bronchial epithelial cells from CFpatients than from healthy subjects, probably as a consequenceof chronic inflammation and infection. This indicates that in CFATP elimination and ADO accumulation on the mucosal surfaceare enhanced (Picher et al., 2004). Mucosal ADO levels reached2-fold higher values in CF epithelial cells. Boucher et al.(Lazarowski et al., 2004) have suggested that ATP but alsoADO could play a key role in regulating ASL volumehomeostasis (see Fig. 2). Indeed, basal ATP levels under restingconditions are insufficient to efficiently activate CaCC and tosustain normal ASL volume (Lazarowski et al., 2004). On theother hand, resting airway epithelia exhibit extracellular ADO

Fig. 2. Extracellular nucleotides modulate the airway surface liquid volume and the mucociliary clearance. (A) Extracellular nucleotides regulate the rates of Na+

absorption and Cl− secretion to adjust PCL volume in non-CF airway epithelia. External ATP activates P2Y2 receptors that trigger Ca2+-dependent Cl− secretion via

CaCC and inhibit Na+ absorption via ENaC. ATP is degraded into ADO by NSAP and ADO activates A2B receptors which stimulate cAMP-dependent Cl− secretionvia CFTR. (B) In CF epithelia, the CFTR defect leads to PCL depletion as a result of a higher basal rate of Na+ absorption, the failure to inhibit Na+ transport rates andthe failure to initiate Cl− secretion via CFTR. In addition the upregulation of NSAP in response to chronic inflammation inhibits the P2Y2-mediated action ofnucleotides on other Cl− and Na+ channels.


concentrations sufficient to activate CFTR (Huang et al., 2001).This activation may involve either A2A or A2B receptors, bothcoupled to cAMP pathway. The effect of ADO on ion transportis mediated by A2B receptors in the mouse trachea (Kornerup etal., 2005), human bronchial epithelial cells (Clancy et al., 1999)and human Calu-3 cells (Huang et al., 2001). The A2A receptoris also expressed by bronchial epithelial cells and seems to beinvolved in the remodeling of the airway epithelium (Allen-Gipson et al., 2006). A role for A1 receptors in airway epithelialion transport has also been proposed (Rugolo et al., 1993;Szkotak et al., 2001). Increased ADO is not expected to improvemucociliary clearance in CF lungs because A2B receptor-mediated stimulation of CFTR channel activity is defective(Boucher, 1994a, 1994b), although an A2B receptor-mediatedactivation of R117H-CFTR has been demonstrated (Clancy etal., 1999). Furthermore, ADOmight have a deleterious effect onNa+ hyperabsorption, since it has been demonstrated that anelevation of cAMP in the CF airway epithelium can enhanceNa+ absorption (Boucher et al., 1986). Since the ADO receptor-CFTR pathway is defective in CF patients, the ASL volumeseems rather dependent on mechanically stimulated ATP release(i.e., coughing, wheezing, and clapping) and CaCC activation(Picher et al., 2004), although that mechanism might becompromised by the upregulation of ectonucleotidases degrad-ing ATP and also UTP.

In addition to stimulating Cl− and water secretion, nucleo-tides can act on the other components of the mucociliaryescalator: mucin secretion and cilia beating. ATP stimulatescilia beating in human nasal epithelial cells via activation of theP2Y2 receptor and via degradation into ADO and activation ofA2B receptors (Wong & Yeates, 1992; Morse et al., 2001). Inhuman nasal epithelial cells, UTP and ATPγS, acted assecretagogues on mucin secretion via Ca2+-dependent pathwaysinvolving P2Y2 and possibly P2Y11 receptors (Chen et al.,

2001; Choi et al., 2003). In addition, UTP and ATP alsostimulate mucin secretion from epithelial goblet cells (Abdullahet al., 1996; Roger et al., 2000). Since CF is characterized byunderhydrated mucus and hypersecretion of mucins (Rose &Voynow, 2006; Voynow et al., 2006) that alter airwaymucociliary clearance, one wonders whether the P2Y-inducedmucin release may be beneficial in CF therapy in promotingmucociliary defense and clearance or rather might contribute topromote airway obstruction.

In summary, there is a complex extracellular nucleotidesignaling at the airway surface involving several ectonucleo-tidases that metabolized nucleotides (Zimmermann, 1996).Thus a large variety of nucleotides and nucleosides arepresent in ASL and may induce diverse and complex cellulareffects by acting on both ADO and nucleotide P2Y and P2Xreceptors.

5. Extracellular nucleotides astherapeutic agents in cystic fibrosis

In CF, defective cAMP-mediated regulation of Cl− con-ductance in AEC, as a result of CFTR mutations, limits thecapacity to secrete Cl−. As a result, CFTR dysfunctions lead to areduced ASL volume with the formation of viscous andunderhydrated mucus that obstructs the CF airways and favorsinfections in CF patients (Fig. 2). However, regulation of Cl−

conductance by other mechanisms, such as the CaCC via a risein intracellular Ca2+, is functional in CF epithelial cells,providing an opportunity to bypass the defective CFTR activity.P2Y nucleotide receptors, notably the P2Y2 subtype, are widelydistributed and have been shown to be prominently localized,like CFTR, to the apical membrane of AEC (Cressman et al.,1999, Homolya et al., 1999). These findings led to the proposalthat ATP or UTP may be used as therapeutic agents for lung


disease in CF epithelium in counterbalancing both the Cl−

secretion defect by activating CaCC and the hyperabsorption ofNa+ by inhibiting ENaC (Knowles et al., 1991; Mason et al.,1991; Mall et al., 2000) and thereby promoting hydration ofairway secretions.

This observation has been made both in vitro, in humannasal epithelial cells, and in vivo: indeed superfusion ofnucleotides, combined to the ENaC blocker amiloride,decreased the nasal transepithelial potential difference bothin normal subjects and in CF patients, whose response hadactually a higher magnitude (Knowles et al., 1991).Furthermore, nucleotides enhance other activities of AECwhich are involved in the mucociliary clearance, such assubmucosal gland secretion and ciliary beating (Bennett et al.,1996). As UTP and ATP were equipotent, pyrimidinenucleotides seem preferable, since they are not degradedinto ADO which induces bronchoconstriction and possiblyairway inflammation (Blackburn et al., 1998; Chunn et al.,2001; for review, see Mohsenin & Blackburn, 2006). A PhaseI study in normal adults demonstrated that aerosolized UTP,alone or combined to amiloride, was safe and enhancedmucociliary clearance (Olivier et al., 1996). In adult CFpatients, aerosolized UTP and amiloride in combinationimproved mucociliary clearance from the peripheral airwaysto near normal values (Bennett et al., 1996). Rapid hydrolysisby ectonucleotidases severely restricts the effectiveness ofnucleotides. The safety of a long-lasting dinucleotide analogof UTP (Up4U, INS365) has been demonstrated in a Phase Istudy involving children and adults with CF (Noone et al.,1999; Kellerman, 2002; Kellerman et al., 2002). dCp4U(INS37217, denufosol tetrasodium), another dinucleotide withan even longer duration of action, has been developedrecently (Yerxa et al., 2002). In AEC or explants, denufosolincreased Cl− and water secretion, cilia beat frequency, andmucin release. The combined effect of these actions wasconfirmed in an animal model of tracheal mucus velocitywhich showed that a single administration of denufosolsignificantly enhanced mucus transport for at least 8 hr afterdosing (Yerxa et al., 2002).

The safety of denufosol was demonstrated in a Phase I/PhaseII trial that involved both adult and pediatric patients with CFwho were administered denufosol or placebo twice daily for5 days (Deterding et al., 2005). In this study, denufosol had noeffect on the lung function, as assessed by the 1-sec forcedexpiratory volume (FEV1), but increased significantly theexpectorated sputum. The results of Phase II clinical trials,funded by the CF Foundation, have been recently reported in apreliminary form. According to Inspire Pharmaceuticals,subjects receiving denufosol (10 mg, 20 mg, 40 mg and60 mg) for 4 weeks had significantly better lung function, asassessed by the FEV1 or forced expiratory flow 25–75%, thanpatients receiving placebo (http://www.inspirepharm.com).Inspire has planned a Phase III program to validate theuse of denufosol as an early intervention therapy for treat-ment of CF patients with mild lung disease (http://www.inspirepharm.com/pipeline.html). Orphan drug status has beengranted to denufosol by both the Food and Drug Adminis-

tration (FDA) and the European Medicines EvaluationAgency (EMEA).

6. Airway inflammation in cystic fibrosisand its potential modulation by nucleotides

Accumulated evidence suggests that early pulmonaryinflammation pathogenesis in CF may be associated with anabnormal increase in the production of pro-inflammatorycytokines in the CF lung, even in the absence of infectiousstimuli (Moss et al., 2000; Stecenko et al., 2001; Puchelle,2002). This dysregulation of cytokine generation by CFepithelial cells has been related with CFTR dysfunction(Moss et al., 1996). Indeed, in CF, production of pro-inflammatory cytokines like IL-1, IL-6, IL-8 or tumornecrosis factor-α was found upregulated whereas IL-10, apotent anti-inflammatory cytokine, was found downregulatedin peripheral blood monocytes as well as in airway cell lines(Bonfield et al., 1995; Tabary et al., 1998). Finally, it hasbeen suggested that a change from Th2 type immuneresponse, most frequent in CF, to a Th1 type immuneresponse might improve the prognosis in CF patients (Moseret al., 2000).

Over the past 15 years, several evidences showed that theimmune system can have profound effect on epithelial function(Berin et al., 1999). The dendritic cells (DC) are immune cellsinitially described in lymphoid organs and having a potentcapacity for presenting an antigen to lymphocytes. DC havebeen recently shown in the normal human lung at the level ofthe bronchioles. The number and state of differentiation of DCvary as a function of the epithelial microenvironment whichseems necessary in the differentiation of DC (Soler et al., 1991).In addition, a very recent study has shown cell–cell interactionbetween DC and epithelial cells (Lambrecht & Hammad, 2003).DC may have a significant role in the pulmonary immuneresponse where they play a pivotal role notably in the initiationof Th2 response-mediated lung inflammation (McWilliam et al.,1995; Masten & Lipscomb, 2000). On the other hand, AEC mayactively influence the activating properties of DC (Rimoldi etal., 2005).

In addition to the regulation of ion transport and othercomponents of mucociliary clearance, nucleotides could havepleiotropic effects on the inflammatory process in CF, both onepithelial cells and infiltrating immune cells. Little is knownabout the effects of nucleotides on the release of cytokines,chemokines, defensins and other proteins involved in theinflammatory reaction by AEC. We have recently investigatedthis question in detail by an approach combining microarrayexperiments, ELISA of specific proteins and chemotaxisexperiments. We show that nucleotides regulate CCL20 andIL-8 release, and thereby modulate immune cells recruitment(Marcet et al., submitted for publication). On the other hand ithas been reported that ATP and UTP promote the secretion ofthe secretory leukocyte protease inhibitor (SLPI) in humantracheal epithelial cells (Merten et al., 1993, 1996, 1998).SLPI is an elastase inhibitor which has anti-inflammatory andantimicrobial properties (Hiemstra, 2002). SLPI may have

http://www.inspirepharm.com

http://www.inspirepharm.com/pipeline.html

http://www.inspirepharm.com/pipeline.html


protective effects, notably in CF airway, by decreasingelastase-dependent lung destruction, and promoting hostdefense against inflammation and infection (Hiemstra, 2002).

The modulation of immune cells by extracellular nucleotideshas been extensively studied in the last years. High concentra-tions of nucleotides, released during tissue injury from activatedplatelets or broken cells, could be important mediators ofinflammation. Recent data indicate that several nucleotide P2Yand P2X receptors are involved in the modulation of theimmune response and can exert pro- or anti-inflammatoryeffects (Fig. 3). Previous studies carried out in our laboratoryhave shown that ATP inhibited the activation of human CD4+ Tlymphocytes (Duhant et al., 2002). It also induced the

Fig. 3. Epithelia intimely interact with immune system by releasing nucleotides and iand inflammatory mediators (cytokines, chemokines) apically and basolaterally thimmature dendritic cells and a subpopulation of T lymphocytes and CCL17/CCLinflammation sites. (B) Extracellular nucleotides also regulate inflammatory propertiIL-10 and IDO production and inhibits release of MCP-1 and MIP-1α from DC. Ainduces chemotaxis and degranulation of neutrophils.

maturation of human DC and inhibited their production ofIL-12 and increased that of IL-10 via activation of the P2Y11

receptor (Schnurr et al., 2000; La Sala et al., 2001; Wilkin etal., 2001, 2002). Furthermore, it has been shown thatextracellular ATP strongly upregulated, in human DC, theexpression of thrombospondin-1 and indoleamine 2,3-dioxy-genase (IDO), 2 mediators involved in immune tolerance(Marteau et al., 2005). These data indicated that extracellularATP, previously considered as a danger signal, could also act as apotent regulator of mediators playing key roles in immunetolerance. In addition, we have recently shown that ATP stronglyreduced the capacity of DC to attract monocytes and immatureDC by inhibiting the release of 2 chemokines: macrophage

nflammatory mediators. (A) Inflamed and infected epithelia release nucleotidesat attract immune cells at inflammation sites. The chemokine CCL20 attracts22 attract B and T cells. The cytokine IL-8 strongly attracts neutrophils ates of immune cells. UDP induces IL-8 release by DC, ATP upregulates TSP-1,TP prevents release of IL-2, IL-5 IL-10 and IFN-γ from T cells. Finally, UTP


inflammatory protein-1alpha (MIP-1α/CCL3) and monocytechemotactic protein-1 (MCP-1) (Horckmans et al., 2006). On theother hand, UTP was known to potentiate neutrophil attractionand degranulation (Verghese et al., 1996; Meshki et al., 2004).Finally, other studies have recently indicated that UDP, themetabolite product of UTP, promoted IL-8 release from DC(Idzko et al., 2004) and monocytes (Warny et al., 2001).

Thus, it could be hypothesized that nucleotides released oraerosolized in the airways could produce effects not only onAEC but also on immune cells such as neutrophils, DC ormacrophages present in inflammation sites. The contribution ofthese actions to the therapeutic efficacy of denufosol remains tobe evaluated.

7. Conclusion

Extracellular nucleotides are signaling molecules that playan important role in airway epithelial homeostasis by mod-ulating ion transport, ASL volume and mucociliary clearance.Based on that physiological role, nucleotides are developed astherapeutic agents for CF. Furthermore extracellular nucleotidesmodulate immune cells function and recruitment and theirrelease by AEC may play a key role in airway inflammation.

Acknowledgments

This work was supported by Research in Brussels, the Fondsde la Recherche Scientifique Médicale (FRSM), the Frenchassociation Vaincre La Mucoviscidose (VLM) and the KingBaudouin Foundation (Fonds Forton). We thank Dr. DidierCommuni for helpful discussion.

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Relationships between cystic fibrosis transmembrane conductance regulator, extracellular nucleotides and cystic fibrosis